1. Field of the Invention
The invention relates to a microassay chip and method for analysis by means of peptides or proteins for use in biological research and biomedical diagnosis.
2. Description of the Related Art
In biological research, biomedicine and industrial applications, large scale genomic evaluation for the detection of specific genes or DNA sequences within a genome, specific gene mutation such as single nucleotide polymorphisms (SNP), and mRNA species are well-established methodologies. These methodologies utilize DNA chips and microarrays on which specific nucleic acid sequences are either synthesized or deposited at individual highly localized positions on an array. These arrays containing the nucleic acid sequences find support on solids such as silicon or glass, or materials such as nylon membranes. The sequences can exist in the array on the order of 103 or 104 individual microsamples because individual “dots” or “pixels” have sub-millimeter characteristic lengths. While these chips have many applications for detecting the presence of and identifying genes in a genome (genotyping) or evaluating patterns of gene regulation (mRNA profiling) in cellular and tissue systems, these nucleic acid-based systems provide no information about the activity or regulation of the gene product, i.e., the synthesized protein.
Currently, DNA chips and microarrays allow genotyping and expression profiling, without rendering information about the activities of enzymes which can be regulated by phosphorylation or cleavage states. Protein chips to date have involved the capture of proteins to immobilized DNA sequences or libraries of immobilized peptides, antibodies or proteins. The three major formats for protein arrays employ plain glass slides, three-dimensional gel pad chips (“matrix” chips) or nanowell chips. None of these formats utilizes soluble substrates to identify numerous enzymes in a simple assay, however.
Proteomic methods typically utilize two-dimensional electrophoresis gels to separate proteins, followed by enzyme digest mapping and/or mass spectrometry to characterize relevant individual proteins in the gel. Neither DNA chips nor two-dimensional electrophoresis provide information about the activity of the protein or its reaction kinetics. For example, an enzyme may require phosphorylation or dephosphorylation in order to have full activity, and prior chip technologies do not provide this information.
Presently, enzyme activity can be measured by incubation of the enzyme with chromogenic substrates whose cleavage products become intensely colored and absorb light at a particular wavelength. Alternatively, the substrate may be a fluorogenic substrate whose cleavage results in leaving groups that are intensely fluorescent when excited at a particular wavelength (λ-EX). Emission wavelengths of the leaving groups may span 10 to 20 nm above and below the maximum λ-EM. This prevents the use of more than two or three different fluorogenic substrates in a single sample to assay for three different enzymatic activities since the emission of each substrate may have significant overlap with the emission of the other substrates. Broad band emission results in color cross-talk and can render false signals. Thus, it is not possible to add 10 to 100 different fluorogenic substrates to a single fluid sample because the emissions would overlap severely. These reactions are typically monitored in cuvettes in a fluorimeter or plate-reader with working volumes of 0.2 to 3 ml. Thus, significant dilution of the sample occurs.
The evaluation of various proteins and/or enzymes within a small biological sample (1.0 to 100 nL) would be useful in analyzing the activity of those proteins and/or enzymes in a number of fields of study. In the field of cell biology and cancer, the timing of cell division is regulated by numerous cyclin-dependent kinases (cdk), cAMP-dependent kinases (PKA), cGMP-dependent kinases (PKG), and calcium-dependent protein kinases (PKC), tyrosine kinases, and tyrosine phosphatases. In the field of hematology, the function of blood is regulated by various coagulation factors, complement factors and fibrinolytic factors which are proteases and inhibitors necessary for thrombotic and thrombolytic mechanisms. During apoptosis programmed cell death) various caspases are critical to the cascade of events. Similarly, neutrophil activation during sepsis, thrombosis or infection is coordinated with release of elastases, proteases or other enzymes. Tumor invasion and intimal hyperplasis can involve the activity of metal metalloproteases (MMPs) and tissue inhibitor of metalloproteases (TIMPs). Various viral activities (e.g., proteases) would be suitable for detection of drug screening of protease inhibitors.
Notwithstanding prior art developments in the areas of peptide and protein chips, therefore, the need for peptide or protein microarrays in diagnostic, prognostic and clinical medicine is large, and largely unmet. Prior art chips do not exist in which a great variety of suspended or soluble chromogenic or fluorogenic substrates may be simply deposited in an array on a support surface, with simple application of the sample fluid thereto for evaluation. At this writing, there are no known peptide or protein chips which can be directly fabricated using a standard contacting or non-contacting microarrayer, for example. Liquid layer sample applications over unbound substrate molecules would be considered unthinkable, moreover, due to the inevitable cross-contamination such liquid sample layers would engender. As a result, a need remains for a simple, effective and inexpensive peptide or protein array or microarray system which provides an easily fabricated chip using standard microarrayer equipment, which provides a system in which elaborate compensations such as peptide or protein binding, or quenching layers are unnecessary, and to which sample may be simply and easily applied. Also, the need likewise persists for a system which can rapidly deliver small liquid samples to individual reactant positions of an array or microarray without cross-contamination among the reactant positions.